The role performed by macrophages during activation of alloreactive cytotoxic T lymphocytes was investigated. Using an experimental approach in which both stimulator and responding populations were depleted of accessory cells, no cytotoxic activity could be induced across H-2 differences. Reconstitution of the response could be achieved using accessory cells of either stimulator or responder origin, but the mechanisms of activation differed fundamentally depending on the H-2 type of macrophages used. Using the lysosomal disruptive drug chloroquine it was found that activation via responder macrophages required antigen processing. In addition this activation pathway was extremely sensitive to blocking by monoclonal anti-Ia antibodies. Reconstitution by stimulator macrophages was both chloroquine insensitive and completely resistant to blocking by anti Ia mAb. We have thus identified two CTL activation pathways (Ia-dependent vs Ia-independent) and demonstrated that macrophages play central yet different roles in initiating these alternative pathways.